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Platelet removal from human blood plasma improves detection of extracellular vesicle-associated miRNA
Jillian W P Bracht1,2,3, Mandy Los1, Monique A J van Eijndhoven2,4
1Amsterdam UMC location University of Amsterdam, Vesicle Observation Centre, Laboratory of Experimental Clinical Chemistry, Department of Clinical Chemistry, Meibergdreef 9, Amsterdam, The Netherlands.
Abstract:
Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery-ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV-miRNA) is affected by the presence of platelets and ery-ghosts. EDTA blood was collected from healthy donors (n = 3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV-miRNA expert lab (non-ISTH protocol). EVs were isolated from plasma by size-exclusion chromatography CL-2B (SEC2B), and concentrations of platelets, activated platelets, ery-ghosts and EVs (150-1000 nm) were measured by calibrated flow cytometry. Two EV-associated miRNAs (let7a-5p and miR-21-5p), and one platelet-associated miRNA (miR-223-3p), were measured by qRT-PCR. Measurements were performed with and without filtration using 0.8 μm track-etched filters to remove platelets and ery-ghosts from plasma and EV-enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery-ghosts, which co-migrated with EVs into the EV-enriched SEC2B fractions. Filtration removed platelets and ery-ghosts (>97%; p ≤ 0.05) and did not affect the EV concentrations (p > 0.17). The miRNA concentrations were 2-4-fold overestimated due to the presence of platelets but not ery-ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV-miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV-miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.
Insights
Filtration of human plasma removes contaminating platelets and erythrocyte ghosts, improving the accuracy of extracellular vesicle microRNA (EV-miRNA) quantification. This step enhances the reliability of EV-miRNA studies by preventing overestimation caused by platelet-associated miRNAs.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Human plasma contains extracellular vesicles (EVs), platelets, and erythrocyte ghosts.
- Accurate quantification of EV-associated microRNAs (EV-miRNAs) is crucial for diagnostic and research applications.
- Contamination by non-EV components can affect EV-miRNA analysis.
Purpose of the Study:
- To investigate the impact of platelets and erythrocyte ghosts on EV-miRNA quantification in human plasma.
- To evaluate the effectiveness of plasma filtration in removing contaminants and improving EV-miRNA analysis.
Main Methods:
- Plasma was prepared using two centrifugation protocols (ISTH and non-ISTH).
- Extracellular vesicles (EVs) were isolated using size-exclusion chromatography (SEC2B).
- Platelets, erythrocyte ghosts, and EVs were quantified by flow cytometry.
- EV-miRNA concentrations were measured by qRT-PCR before and after filtration with 0.8 μm track-etched filters.
Main Results:
- Both centrifugation protocols yielded plasma containing platelets and erythrocyte ghosts that co-migrated with EVs.
- Filtration effectively removed >97% of platelets and erythrocyte ghosts without altering EV concentrations.
- Platelet presence led to a 2-4 fold overestimation of miRNA concentrations, while erythrocyte ghosts did not significantly impact results.
- Filtration significantly improved the accuracy of EV-miRNA measurements.
Conclusions:
- Platelets, but not erythrocyte ghosts, significantly overestimate EV-miRNA concentrations in human plasma.
- Filtration of human plasma is a critical step for enhancing the comparability and reproducibility of quantitative EV-miRNA studies.
- Recommendations include measuring plasma platelet concentration and filtering plasma before downstream analyses or biobanking.
